Process for producing amide compound using microbial catalyst

ABSTRACT

This invention relates to a process for producing an amide compound from a nitrile compound using a microbial catalyst, wherein a microbial cell having nitrile hydratase activity of 50 U or higher per mg of dry cell at a reaction temperature of 10° C. is brought into contact with a nitrile compound in an aqueous medium without being immobilized. This method utilizes a microbial cell that exhibits high nitrile hydratase activity in the reaction without being entrap-immobilized. Thus, an amide compound can be effectively produced from a nitrile compound without problems of decreased reaction speed or lowered amount produced per unit cell amount, which are caused by entrap-immobilization. Accordingly, an amide compound can be produced within a very short period of time in the case of a batch reaction and with a very small-scale facility in the case of a continuous reaction.

This application is a national stage entry of PCT/JP01/11149, filed Dec.19, 2001 which claims priority to Japanese Application No. 2000-387537,filed Dec. 20, 2000.

TECHNICAL FIELD

The present invention relates to a process for producing an amidecompound from a nitrile compound using a microorganism having nitrilehydratase activity.

BACKGROUND ART

Recently, a process for synthesizing a compound using a biocatalyst hasbeen used for producing a variety of compounds because of advantagessuch as moderate reaction conditions, simplified reaction processes, andhigh purity of reaction products due to small amounts of by-products.

Since the discovery of nitrile hydratase, an enzyme that converts anitrile compound into an amide compound, biocatalyst utilization hasbeen actively studied in the production of amide compounds (JP PatentPublication (Kokai) No. 11-123098, JP Patent Publication (Kokai) No.7-265091, JP Patent Publication (Kokoku) No. 56-38118, and JP PatentPublication (Kokai) No. 11-89575).

At present, a microorganism having nitrile hydratase activity is usedfor producing acrylamide, nicotineamide, or the like at the industriallevel for a superior reaction process from the viewpoints ofoperability, safety, economic efficiency, and other factors.

Up to the present, a considerable number of microorganisms have beenfound having nitrile hydratase activities. Examples thereof includemicroorganisms belonging to the genera Nocardia, Corynebacterium,Bacillus, Pseudomonas, Micrococcus, Rhodococcus, Acinetobacter,Xanthobacter, Streptomyces, Rhizobium, Klebsiella, Enterobacter,Erwinia, Aeromonas, Citrobacter, Achromobacter, Agrobacterium, andPseudonocardia.

Among them, the genera Pseudomonas, Bacillus, Rhodococcus, andPseudonocardia express a nitrile hydratase having a very high level ofactivity and stability. Thus, they are used at the industrial level orlevels similar thereto.

Further, when culturing these microorganisms, it is known that amicrobial cell having highly active nitrile hydratase is obtained by amethod of adding nitriles or amides (JP Patent Publication (Kokoku) Nos.61-43996 and 61-43999), a method of adding amino acid (JP PatentPublication (Kokoku) Nos. 61-43997 and 61-43998), a method of addingsome kind of metal ion (JP Patent Publication (Kokai) No. 61-162193, JPPatent Publication (Kokoku) No. 6-55148, and JP Patent Publication(Kokai) No. 8-187092), or the like.

In contrast, a biocatalyst has low stability with regard to heat, andthus, reactions must be carried out at low temperatures. This results ina decreased reaction speed per catalyst. When producing a compound usinga biocatalyst at the industrial level, therefore, the catalystconcentration in the reaction tank should be raised.

A currently known industrial process for producing an amide compoundfrom a nitrile compound using a biocatalyst is similarly carried out byimmobilizing microbial cells to make them particulate, raising thecatalyst concentration in the reaction tank, and facilitating catalystseparation (see Kagaku to Kogyo (Chemistry and Chemical Industry) Vol.43, No. 7, p. 1098-1101 (1990), JP Patent Publication (Kokai) Nos.54-143593 and 54-144889). Also, a method for immobilizing the cell isstudied (see JP Patent Publication (Kokai) Nos. 57-39792 and 62-294083).When effective production of an amide compound at the industrial levelis intended, immobilization of cells at a higher concentration has beenconsidered to be important (see JP Patent Publication (Kokai) No.7-203964).

However, the present inventors entrap-immobilized a microbial cell thatexhibits high nitrile hydratase activity and used it in the reaction. Asa result, it was confirmed that a nitrile compound as a reactionsubstrate and/or an amide compound as a reaction product causeddiffusion defects in the entrap-immobilized particles, and the reactionspeed was significantly decreased.

For example, according to the comparison of initial reaction speedsbetween the entrap-immobilized cell and the unimmobilized cell, thereaction speed was significantly decreased to one-tenth or lower,depending on reaction conditions. Not only the initial reaction speed issignificantly decreased, but also the activity of the enzyme in theentrap-immobilized catalyst, which does not fully contribute to thereaction due to diffusion defect, is lowered during the reaction. Thisalso lowers the amount of amide compound produced per unit cell amount.

Specifically, decreased reaction speed or lowered amount of amidecompound produced per unit cell amount as mentioned above results inunfavorable conditions. Under such conditions, it takes a long time toaccumulate the targeted amount when producing an amide compound by abatch reaction, and the size of facilities must be enlarged in the caseof a continuous reaction.

Accordingly, an object of the present invention is to solve problemsoccurring in the process for producing an amide compound from a nitrilecompound using a biocatalyst, such as decreased reaction speed orlowered amount of amide compound produced per unit cell amount. Theseproblems are caused by the use of immobilized microbial cell with highlyexhibited nitrile hydratase activity.

In order to attain the above object, the present inventors haveconducted concentrated studies concerning a more suitable form ofcatalyst than the immobilized catalyst. As a result, they have foundthat an amide compound could be more effectively produced when using amicrobial cell that exhibits high nitrile hydratase activity of 50 U orhigher per mg of dry cell at 10° C., and bringing the microbial cellinto contact with a nitrite compound while suspended in an aqueousmedium, than is the case when immobilizing the cell. This has led to thecompletion of the present invention.

More specifically, the present invention relates to a process forproducing an amide compound from a nitrile compound using a microbialcatalyst, wherein a microbial cell having nitrile hydratase activity of50 U or higher per mg of dry cell at a reaction temperature of 10° C. isbrought into contact with a nitrile compound in an aqueous mediumwithout being immobilized.

DISCLOSURE OF THE INVENTION

The present invention is hereafter described in detail.

In the present invention, any microorganisms may be used as long as theyhave nitrile hydratase activity of 50 U or higher per mg of dry cell ata reaction temperature of 10° C. Examples of preferable microorganismsinclude those belonging to the genera Bacillus, Bacteridium,Micrococcus, Brevibacterium (JP Patent Publication (Kokoku) No.62-21519), Corynebacterium, Nocardia (JP Patent Publication (Kokoku) No.56-17918), Pseudomonas (JP Patent Publication (Kokoku) No. 59-37951),Microbacterium (JP Patent Publication (Kokoku) No. 4-4873), Rhodococcus(JP Patent Publication (Kokoku) Nos. 4-4873, 6-55148, and 7-40948),Achromobacter (JP Patent Publication (Kokai) No. 6-225780), andPseudonocardia (JP Patent Publication (Kokai) No. 9-275978). Bacteria ofthe genus Rhodococcus are more preferable.

Alternatively, a transformant may be used. This is prepared by obtainingthe aforementioned microorganism-derived nitrile hydratase gene andintroducing the gene as such, or an artificially modified form thereof,into an arbitrary host.

Examples of the transformants include E. coli MT 10770 (FERM P-14756)transformed with a nitrile hydratase of the genus Achromobacter (JPPatent Publication (Kokai) No. 8-266277), E. coli MT 10822 (FERMBP-5785) transformed with a nitrile hydratase of the genusPseudonocardia (JP Patent Publication (Kokai) No. 9-275978), or amicroorganism transformed with a nitrile hydratase of the genusRhodococcus rhodochrous (JP Patent Publication (Kokai) No. 4-211379).

The unit “U” of enzyme activity used in the present invention means that1 μmol of corresponding amide compound is generated from a nitrilecompound per minute. The term “enzyme activity” used herein refers tothe value of enzyme activity measured utilizing a nitrile compound thatis used in production.

The enzyme activity is measured by placing 5 mL of 50 mM phosphatebuffer adjusted to the optimal pH (e.g., pH 7) of the enzyme in a testtube having a diameter of 30 mm, suspending 2 mg of the cultured andwashed cells (dry weight) therein, and shaking the tube in a water tankat 10° C. About 5 minutes later, a phosphate buffer prepared in advancecontaining 1 to 5% of nitrile compound, placed at 10° C. and adjusted tothe optimal pH, is added. The concentration of the amide compoundgenerated after an arbitrary reaction time is measured using analyzingequipment such as gas chromatography or liquid chromatography, therebycalculating the enzyme activity.

The reaction time is determined in such a manner that a reactionsolution retains a nitrile compound of a concentration at which thereaction speed is not decreased, and the concentration of the amidecompound generated is high enough to be accurately measured at the endof the reaction.

The present invention is effective when a microbial cell having enzymeactivity of 50 U or higher per mg of dry cell at 10° C. is used. It ismore effective with the use of a microbial cell having enzyme activityof 80 U or higher, and even more effective with activity of 100 U orhigher.

The nitrile compound according to the present invention is convertedinto a corresponding amide compound through the action of a nitrilehydratase. Examples thereof include: aliphatic saturated nitriles asexemplified by acetonitrile, propionitrile, succinonitrile, andadiponitrile; aliphatic unsaturated nitriles as exemplified byacrylonitrile and methacrylonitrile; aromatic nitriles as exemplified bybenzonitrile and phthalodinitrile; and heterocyclic nitriles asexemplified by 3-cyanopyridine and 2-cyanopyridine. Because of thechemical and physical properties of a nitrile compound, the substratespecificity of a nitrile hydratase enzyme, and the industrial point ofview, acrylonitrile and cyanopyridine are preferable as target compoundsof the present invention.

In the present invention, a form of catalyst without beingentrap-immobilized is such that a membrane of a microbial cell is in adirect contact with a reaction solution. An example thereof is a form ofcatalyst treated in an entrap-immobilization method in which a cell isnot entrapped with high molecular substances such as polyacrylamide,polyvinyl alcohol, carrageenan, agar, gelatin, or alginic acid.

More specifically, the “catalyst without being entrap-immobilized”according to the present invention is a microbial cell itself that wascultured and optionally subjected to washing or other forms oftreatment, a microbial cell that was chemically treated with a substancehaving a polyfunctional group such as glutaraldehyde, or a microbialcell that was chemically bonded onto a surface of a glass bead, resin,silica gel, or the like.

The use of a cell chemically treated with glutaraldehyde as a catalystis particularly preferable from the viewpoint of improvement in thestability of catalyst enzyme activity.

The operation that brings a microbial cell into contact with a nitrilecompound in an aqueous medium refers to one in which a microbial cellhaving nitrile hydratase activity is brought into contact with a nitrilecompound in water or in an aqueous medium prepared by dissolving, forexample, a stabilizer for ion strength, pH buffer capacity, or nitrilehydratase activity in water. This may be carried out by a batch systemor a continuous system. A form of reaction is selected depending onproperties of reaction substrate, reaction solution, target compound,and the like, or scale of production, and a reaction apparatus isdesigned based thereon.

Preferably, reaction conditions such as reaction temperature and pH arecontrolled to be optimal so that an amide compound can be produced on asmaller scale or within a shorter time.

The concentration of the amide compound accumulated by the above methodis preferably 20% or higher, and more preferably 50% or higher, from anindustrial point of view.

This description includes part or all of the content as disclosed in thedescription of Japanese Patent Application No. 2000-387537, which is apriority document of the present application.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is hereafter described in more detail withreference to the following examples, although it is not limited thereto.In the following examples, “%” is by mass unless otherwise specified.

Example 1 Production of Acrylamide Using a Microbial Cell in an AqueousMedium

(1) Culture of Cell

(i) Conditions for Preculture:

(Composition of Medium)

2% fructose, 5% polypeptone (Nihon Pharmaceutical Co., Ltd.), 0.3% yeastextract (Oriental Yeast Co., Ltd.), 0.1% KH₂PO₄, 0.1% K₂HPO₄, 0.1%MgSO₄.7H₂O, pH 7

(Culture Method)

A medium (100 ml) was fractionated into a 500 ml conical flask, theflask was cotton plugged, and it was then sterilized in an autoclave at121° C. for 20 minutes. Rhodococcus rhodochrous J1 (FERM BP-1478) wasinoculated and subjected to shake culture at 30° C. for 48 hours.

(ii) Conditions for Main Culture:

(Composition of Medium)

-   Initial medium: 0.2% yeast extract, 0.1% KH₂PO₄, 0.1% K₂HPO₄, 0.1%    MgSO₄.7H₂O, 0.002% CoCl₂.6H₂O, 0.025% ammonium sulfate, 2% fructose,    2% urea, 0.4% ethanol, 0.1% Pluronic L61 (Asahi Denka Co., Ltd.), pH    7-   Medium added later: 20% fructose, 5% ethanol, 6% ammonium sulfate,    pH 6.5    (Culture Method)

The initial medium (2 liters) was fractionated into a 3-liter mini-jarfermenter and sterilized in an autoclave at 121° C. for 20 minutes.Separately, fructose, ethanol, and urea were aseptically filtered usinga 0.45 micron filter paper (Advantec Toyo Kaisha, Ltd.) and added to themedium.

Culture was conducted under conditions at a pressure inside the tank of0.098 MPa, an agitation rate of 600 rpm, an air-flow rate of 1 vvm, a pHof 7, and a temperature of 30° C. Culture was terminated when themaximal enzyme activity occurred. Thereafter, the culture product waswashed with a 50 mM phosphate buffer (pH 7.7), and a suspension of cell(weight of dry cell: 15%) was obtained.

(2) Measurement of Nitrile Hydratase Activity

A 50 mM phosphate buffer (4.98 ml, pH 7.7) and 20 μL of suspension ofcell were added and mixed in a test tube having a diameter of 30 mm, andthe tube was shaken in a tank at 10° C. for 5 minutes. A 50 mM phosphatebuffer (5 mL, pH 7.7) containing 5.0% acrylonitrile, which waspreviously set at 10° C., was added thereto, the product was allowed toreact for 10 minutes, the cells were separated by filtration, andacrylamide generated was quantified by gas chromatography (GC-14B,SHIMADZU CORPORATION). Analysis was conducted using a 1 m glass columnfilled with Parabox PS (a column filler, Waters) at a column temperatureof 230° C., and the FID detection was conducted at 250° C. The resultindicated that 1.2% of acrylamide was generated. When “1 U” is definedas an amount of activity resulting when 1 micromole of acrylonitrile isconverted into acrylamide at a reaction temperature of 10° C. within areaction time of 1 minute, the activity of the cell for convertingacrylonitrile into acrylamide was 56 U per mg of dry cell at 10° C.

(3) Conversion of Acrylonitrile into Acrylamide

A 50 mM TRIS (2-amino-2-hydroxymethyl-1,3-propanediol) hydrochloridebuffer (664 g, pH 7.7) was placed in a 1 liter-jacketed separable flask.The suspension of cell obtained above was added thereto so as to bringthe weight of dry cell to 90 mg. Acrylonitrile was continuously addedthereto in order to maintain the acrylonitrile concentration at 2% at anagitation rate of 180 rpm at 18° C.

As a result, the concentration of the acrylamide, which was produced 25hours after the initiation of acrylonitrile addition, reached the targetlevel of 45%.

Comparative Example 1 Production of Acrylamide Using anEntrap-Immobilized Microbial Cell

(1) Immobilization of Cells

The suspension of cell obtained in Example 1 having activity of 56 U interms of converting acrylonitrile into acrylamide was added to anequivalent amount of an aqueous solution of thoroughly dissolved 3%sodium alginate (Kanto Kagaku), and they were thoroughly mixed. Thismixture was added dropwise to an aqueous solution of 1M calcium chloridethrough a silicon tube having an inner diameter of 2 mm. Thus, particlesof immobilized cell having particle diameters of about 3 mm wereobtained. The particles of immobilized cell were washed with a 50 mMTRIS hydrochloride buffer (adjusted to pH 7.7) to obtain immobilizedcells.

(2) Conversion of Acrylonitrile into Acrylamide

A 50 mM TRIS hydrochloride buffer (664 g, pH 7.7) was placed in a 1liter-jacketed separable flask. The immobilized cells obtained abovewere added thereto so as to bring the weight of dry cell to 90 mg.Acrylonitrile was continuously added thereto in order to maintain theacrylonitrile concentration at 2% at an agitation rate of 180 rpm at 18°C.

As a result, the acrylamide concentration did not reach the target levelof 45% even 50 hours after the initiation of acrylonitrile addition.

Example 2 Production of Acrylamide Using Microbial Cell in an AqueousMedium

(1) Culture of Cell

Pseudomonas chlororaphis B23 (FERM BP-187) cell was cultured in themanner as described in the Example of JP Patent Publication (Kokai) No.2-177883. The activity of this cell for converting acrylonitrile intoacrylamide was measured in the same manner as in Example 1 at pH 7.7. Asa result, the activity was 90 U per mg of dry cell at 10° C.

(2) Conversion of Acrylonitrile into Acrylamide

A 50 mM phosphate buffer (850 mL, pH 7.7) and 0.4 g of cell (on a drybasis) were added to a jacketed separable flask (internal volume: 1liter). The reaction was carried out by continuously addingacrylonitrile while stirring at 3° C. to maintain the acrylonitrileconcentration at 2%.

The acrylamide concentration reached the target level of 20% three hourslater.

Comparative Example 2 Production of Acrylamide using Entrap-ImmobilizedMicrobial Cell

(1) Entrap-Immobilization of Cell

An aqueous solution of monomer mixture was prepared so as to comprise30%, 1%, and 4% of acrylamide, methylenebisacrylamide, and2-dimethylaminopropyl methacrylamide, respectively.

Subsequently, the suspension of cell having activity of 90 U in terms ofconverting acrylonitrile into acrylamide obtained in Example 2, anaqueous monomer solution, an aqueous solution of 10%N,N,N′,N′-tetramethyl ethylene diamine, and an aqueous solution of 10%ammonium persulfate were subjected to line-mixing at a mixing ratio of50:20:1:1. The effluents were successively placed on a bat having a sizeof 300×300×30 mm and then polymerized thereon.

The produced cell-immobilized gel sheet was cut into small pieces ofabout 0.5 mm² using a knife to obtain particles of acrylamide polymerentrap-immobilized cell. The particles of immobilized cell were washedby dipping in an aqueous solution of 0.1% sodium acrylate (adjusted topH 7 with the aid of sodium hydroxide) for preparation.

(2) Conversion of Acrylonitrile into Acrylamide

Acrylonitrile was converted into acrylamide using the method and theapparatus as described in Example 2.

The acrylamide concentration did not reach the target level of 20% eighthours later.

Comparative Example 3 Production of Acrylamide using a Microbial CellHaving Low Nitrile Hydratase Activity

(1) Culture of Cell and Preparation of Catalyst

In the same manner as in Example 1, the Rhodococcus rhodochrous J1 (FERMBP-1478) cell was cultured. When the activity of the cell for convertingacrylonitrile into acrylamide, which was measured based on the methodfor measuring activity as described in Example 1, reached 20 U per mg ofdry cell at 10° C., culture was terminated. Thereafter, the cultureproduct was washed with a 50 mM phosphate buffer (pH 7.7), and asuspension of cell (weight of dry cell: 15%) was obtained.

(2) Conversion of Acrylonitrile into Acrylamide

In accordance with the method described in Comparative Example 2, asuspension of acrylamide polymer entrap-immobilized cell was firstprepared.

Subsequently, the aforementioned suspension of immobilized cell orsuspension of unimmobilized cell was used as a microorganism in anamount of 225 mg in terms of the weight of dry cell, thereby convertingacrylonitrile into acrylyamide. As a result, the acrylamideconcentration reached the target level of 45% about 100 hours later withthe use of a suspension of either immobilized or unimmobilized cells.

Specifically, there was no significant difference between immobilizedcells and unimmobilized cells when the cell exhibited activity of 20 U.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

In the method according to the present invention, a microbial cell thatexhibits high nitrile hydratase activity is used in the reaction withoutbeing entrap-immobilized. Thus, an amide compound can be effectivelyproduced from a nitrile compound without problems of decreased reactionspeed or lower amount produced per unit cell amount, which are caused byentrap-immobilization. Accordingly, an amide compound can be producedwithin a very short period of time in the case of a batch reaction andwith a very small-scale facility in the case of a continuous reaction.

1. A process for producing an amide compound from a nitrile compoundcomprising: contacting an aqueous medium of acrylonitrile orcyanopyridine with a suspension of microbial cells having nitrilehydratase activity of at least 50 U per mg of dry cell measured at areaction temperature of 10° C. for a time and under conditions suitablefor conversion of acrylonitrile or cyanopyridine into its correspondingamide compound, continuously adding the acrylonitrile or cyanopyridineto the aqueous medium, and obtaining a concentration of at least 20% ofthe amide compound in the aqueous medium; wherein said microbial cellshaving nitrile hydratase activity are at least one of Rhodococcusrhodochrous J1 (FERM BP-1478) and Pseudomonas chlororaphis B23 (FERMBP-187).
 2. A process for producing an aliphatic saturated amide, anaromatic amide, or a heterocyclic amide from a substrate that is analiphatic saturated nitrile, an aromatic nitrile, or a heterocyclicnitrile, respectively, comprising: contacting a suspension of microbialcells having nitrile hydratase activity of at least 50 U per mg of drycell measured at a reaction temperature of 10° C. with an aqueousreaction solution containing an aliphatic saturated nitrile, an aromaticnitrile, or a heterocyclic nitrile for a time and under conditionssuitable for the production of aliphatic saturated amide, an aromaticamide, or a heterocyclic amide, continuously adding the aliphaticsaturated nitrile, an aromatic nitrile, or a heterocyclic nitrile to theaqueous reaction solution, and recovering a concentration of at least20% of the aliphatic saturated amide, the aromatic amide, or theheterocyclic amide in the aqueous reaction solution, wherein saidmicrobial cells having nitrile hydratase activity are at least one ofRhodococcus rhodochrous J1 (FERM BP-1478) and Pseudomonas chlororaphisB23 (FERM BP-187).
 3. The process of claim 2, wherein said substrate isacrylonitrile.
 4. The process of claim 2, wherein said substrate iscyanopyridine.